Hormone supplemented defined media able to support the growth and differentiation of several embryonal carcinoma (EC) cell lines have been developed in order to identify extracellular signals that induce differentiation. Previously a defined medium (EM-3) containing fibronectin, insulin and transferrin was developed for the growth of the EC cell line F9. In EM-3 Fg, cells undergo little or no differentiation unless an inducer such as retinoic acid is added. In direct contrast to these results at least two other EC cell lines spontaneously differentiate when transferred from serum containing media to defined media. The cells formed are similar in two respects to those formed when F9 are exposed to retinoic acid: their morphology and the secretion of plasminogen activator. These results strongly suggest that a factor(s), present in serum, blocks the differentiation of some, but not other, EC cell lines. In related studies the growth requirements of F9 lines and their differentiated cells were examined further. Epidermal growth factor and sarcoma growth factor were found to stimulate the growth of the differentiated cells but not that of the parent cells. Growth of F9 at low density was found to require the addition of lipoproteins to EM-3.